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Abstract

Although yeast two-hybrid experiments are commonly used to identify protein interactions,
the frequent occurrence of false negatives and false positives hampers data interpretation.
Using both yeast one-hybrid and two-hybrid experiments, we have identified potential
sources of these problems: the media preparation protocol and the source of the yeast
nitrogen base may not only impact signal range but also effect whether a result appears
positive or negative. While altering media preparation may optimize signal differences
for individual experiments, media preparation must be reported in detail to replicate
studies and accurately compare results from different experiments.

Keywords:

Findings

The yeast two-hybrid system provides an efficient method for identifying novel protein·protein
interactions in small-scale studies and high-throughput screens [1-3]. In this system, the first hybrid is composed of a bait protein fused to a DNA binding
protein that recognizes DNA sequences upstream of a lacZ reporter gene. The second hybrid protein consists of a strong activation domain fused
to a potential protein partner. Interaction between bait and partner proteins localizes
the strong activation domain to the lacZ promoter, thus activating transcription of the lacZ reporter gene. Multiple cycles of lacZ transcription, translation, and β-galactosidase cleavage of X-gal generate visible
quantities of the blue assay product, 5-bromo-4-chloro-3-hydroxyindole. This sensitive
system detects interactions that may be difficult to observe by other means (e.g.,
co-immunoprecipitation) due to low abundance of the protein complex.

The sensitivity of this method can also lead to several problems: (i) transformation
efficiency affects signal strength and is highly dependent on technique [3], (ii) 25% to 50% of detected interactions are estimated to be false positives [2,4,5], and (iii) 55% to 90% of true protein interactions are not observed (false negatives)
[2,5]. Furthermore, differences in protein abundance can visibly impact outcome [6,7]. Given these difficulties, it is perhaps not surprising that laboratories using similar
assays report different protein interactions [2,5].

Identifying sensitive parameters in yeast two-hybrid experiments could help address
these difficulties. Herein, we report both the source of media components and the
media preparation protocol can impact yeast one-hybrid and two-hybrid results. In
the yeast one-hybrid system, the bait protein, which includes a transcription activation
domain, is fused to a DNA binding protein [1]. When the protein of interest is hybridized to a DNA binding domain, this assay can
be used to assess the presence and strength of an activation domain [8]. Alternately, the protein of interest can be hybridized to a strong activation domain
to screen for DNA binding [9]. The yeast one-hybrid method requires transformation of only one plasmid, resulting
in improved transformation efficiency and fewer cellular manipulations [3]. This simpler experimental design reduces experimental variability, allowing focus
on systematic issues that arise using this method.

Experimental Design

Media were prepared according to the protocol in Additional File 1 with varying parameters as discussed below. EGY48-p8op-lacZ yeast were transformed
as previously described [4,8,10]. The genome of this reporter host strain (EGY48) includes a wild-type LEU2 gene under
the control of a series of LexA operators. The reporter plasmid p8op-lacZ, which carries
the lacZ gene under the control of LexA operators, had been stably transformed into
this strain [8]. For each DNA tested, cells from a single transformation were divided between plates
for each condition to ensure that commonly cited error sources, such as interactions
with endogenous proteins, autoactivation, and low transformation efficiencies [1-5], did not contribute to differences in results. The resulting blue color of yeast
colonies, which reflects the level of transcription activation, was gauged with a
standard color chart (Additional File 2). Analysis by color chart yielded data comparable to analysis by enzyme assay. All
experiments were repeated at least three times and yielded consistent results. Yeast
nitrogen base sources were Sigma (Y1251-100G), Difco (233520), and Clontech (630421).

Effects of Yeast Nitrogen Base Source on Yeast-One Hybrid Experiments

We first compared media in which the yeast nitrogen base (YNB) was produced by different
companies and the pH was adjusted after sterilization to ensure consistency between
media preparations. In general, media made with YNB from Sigma yielded the strongest
signal in yeast one-hybrid experiments, whereas media made with Difco YNB yielded
intermediate results, and media made with Clontech YNB produced the weakest signal
(Table 1 Conditions 1-3). We began experiments using pLexA-Gal4, in which DNA encoding the
LexA DNA binding domain is fused to DNA for the Gal4 transcription activation domain.
Expression of this hybrid protein should produce a strong positive signal. At only
48 hours post-transformation, yeast colonies transformed with pLexA-Gal4 appeared
light blue on media made with Sigma and Difco, whereas colonies on the Clontech media
remained white. After 60 hours post-transformation, pLexA-Gal4 colonies on Sigma and
Difco media were medium blue, and the colonies on the Clontech media were light blue.
Although the results from Difco and Clontech media became indistinguishable after
72 hours, colonies on the Sigma plates were still darker blue. While the source of
YNB made a consistent difference, using different lots from the same supplier had
no impact on results (Additional File 3).

To determine whether this media effect is unique to LexA-Gal4, we next tested LexA-Ubx,
in which LexA is fused to the Drosophila Hox protein Ultrabithorax (Ubx), which activates transcription at a moderate level
to yield medium blue colonies [8]. Colonies transformed with pLexA-Ubx yielded a similar trend as pLexA-Gal4: colonies
on media containing Sigma and Difco YNB were the first to show blue color (Table 1 Conditions 1-3).

As a negative control, we used LexA-Pro2 in which LexA is fused to a Ubx variant containing
mutations A223P/Q224P/T225P/A228P, which disrupt a predicted α-helix required for
transcription activation [8]. Importantly, these proline mutants also prevent transcription activation by Ubx
in a promoter-reporter assay in Drosophila S2 cells, corroborating the yeast results [8]. Whereas we expected colonies expressing LexA-Pro2 to be white, colonies grown on
all three types of media were light blue after 72 hours. Consequently, the sensitivity
of the signal to changes in media appears to depend on the strength of transcription
activation: the strongest activator, LexA-Gal4, generated the largest differences,
whereas results for the weakest activator, Pro2, were independent of YNB source for
this media preparation method.

Based on our prior results, we expected to see a wide range of color: dark blue (++++)
for LexA-Gal4, medium blue (+++) for LexA-Ubx, and white (-) for LexA-Pro2 [8]. However, the results for wild-type and mutant Ubx were similar at 72 hours post-transformation
in all three conditions. Furthermore, colonies expressing LexA-Gal4 were lighter than
expected when grown on Clontech and Difco media. Thus, the range of signal strengths
appears poor for Conditions 1-3 (Table 1).

Effects of Adjusting Media pH on Yeast-One Hybrid Experiments

Strikingly, simply not readjusting the pH after sterilization resulted in amplification
of the signal range. Comparing results for wild-type and mutant Ubx, a slight difference
in signal was again observed in Sigma media which persisted to 72 hours (Table 1, Condition 4). However, using Difco YNB without pH re-adjustment not only increased
the signal range at 72 hours but also matched prior results in yeast and Drosophila cell culture [8]. Specifically, for Condition 3 (Difco, no pH adjustment), the data ranged from light
blue (++, Ubx and Pro-2) to medium blue (+++, Gal4), whereas for Condition 6 (Difco,
pH adjustment), the data ranged from white (-, Pro-2) to dark blue (++++, Gal4).

Even without readjusting the pH, media using YNB sources did not replicate previous
data in yeast and Drosophila cell culture. Consequently, media choice can influence the prevalence of false positives
and false negatives. Expression of the LexA-Gal4 positive control correctly yielded
a strong positive signal at 48 hours on Sigma media, but a false negative signal on
Difco media. Likewise, although LexA-Ubx expression rapidly generates a positive signal
on Sigma plates, expression of LexA-Ubx on Difco plates gives a false negative response
at 60 hours post-transformation, although a positive signal eventually develops after
longer incubations. Consequently, the researcher could miss this signal, since the
pLexA-Gal4 positive control produces signal at 60 hours. Conversely, the Ubx mutant
Pro-2, which should not activate transcription, yields a false positive signal on Sigma media at after 60
hours incubation. Therefore, by not re-adjusting the pH after autoclaving, we observed
a greater overall range of signal, greater media-dependent differences in signal,
and greater differences in signal between transcription activators of different strengths.

Media Effects on Yeast-Two Hybrid Experiments

Given that the source of the YNB had a profound impact on the results observed in
a yeast one-hybrid assay, we reasoned that differences in media might partially account
for the variations in data originating from different laboratories in yeast two-hybrid
experiments [2,3]. Testing this possibility, we examined the impact of the media source in yeast two-hybrid
experiments using a set of control plasmids, in which the T-Antigen-B42 activator
chimera should bind a LexA-p53 chimera but not a LexA-Lamin chimera. Similar to yeast
one-hybrid experiments, we found the results of yeast two-hybrid data were also dependent
on media (Table 2). At five days after transformation, only media produced with Clontech YNB yielded
a positive signal, and at six days after transformation, colonies on Clontech media
had the strongest signal and Sigma media consistently yielded the weakest signal.
Surprisingly, this trend is opposite that observed for yeast one-hybrid experiments,
suggesting either the two experimental methods are differentially sensitive, or the
magnitude of the media effect is influenced by the proteins assayed.

In summary, we have demonstrated that i) media components and preparation methods
influence yeast one- and two-hybrid experiments, ii) for yeast one-hybrid experiments,
this media sensitivity scales with increasing activation domain strength, and iii)
media can determine the range of signal observed in yeast lacZ reporter assays. Such differences are sufficient to change data interpretation: experiments
that should yield a negative result may appear positive, or vice versa. We observed
media-dependent effects in four systems: yeast one-hybrid experiments using either
the Gal4 activation domain, wild-type Ubx, or a Ubx mutant lacking activity, and finally
yeast two-hybrid experiments reporting on the p53·T-antigen interaction. Therefore,
this media sensitivity occurs across a wide range of yeast experiments and may contribute
to differences in yeast two-hybrid results observed previously [2,11]. Our results for a single yeast nitrogen base source are consistent even when testing
different lot numbers or chemicals differing in age by a decade, suggesting that the
differences we observe when the source of the yeast nitrogen base is changed are significant.
Since each manufacturer reports the same yeast nitrogen base composition, these differences
may be attributed to differences in contaminants of these media components (Table
1). These experiments suggest that careful reporting of chemical sources and preparation
methods for yeast media is essential to enable replication of experiments and comparison
of data between studies. Finally, these media-dependent differences in results could
be exploited to enhance and thus verify weak differences in signals from yeast assays.

Competing interests Statement

The authors declare that they have no competing interests.

Authors' contributions

YL discovered the media effect on yeast one- and two-hybrid results. ZM, HCH, and
KG collected the remaining data and edited the text. KM and SB wrote and edited the
text. All authors have read and approved the final manuscript.

Acknowledgements

The authors would like to thank Dr. Xin-Xing Tan for many of the plasmids used in
these experiments. This research was supported by grants from the Robert A. Welch
Foundation (C-576) and National Institutes of Health (GM22441) to K.S.M. and from
the Texas A&M Health Science Center to S.E.B.